Alterations in serotonin (5-hydroxytryptamine, 5-HT) neurotransmission have long been theorized to play an important role in the pathogenesis of psychiatric disorders, particularly depression. This theory stems from the facts that drugs (e.g., SSRIs) increasing levels of extracellular 5-HT (5-HTExt) treat depression with moderate efficacy and that anomalies in putative biomarkers of central 5-HT function have been repeatedly reported in depression. However, brain 5-HT dysfunction has never been directly demonstrated in depression patients and whether low 5-HT can elicit or predispose to depression remains unclear. The rate-limiting step in brain 5-HT synthesis is the conversion of tryptophan to 5-hydroxytryptophan (5-HTP) by tryptophan hydroxylase 2 (Tph2). The recent identification of functional mutations in several of the genes involved in 5-HT homeostasis, including tph2, and their associations with depression or impaired therapeutic responses to SSRIs have stimulated renewed interest in the 5-HT deficiency theory of depression. We have generated a mouse line carrying a rare Tph2(R439H) mutation originally identified in a depression cohort. In these mutant mice brain 5- HT synthesis and tissue storage are decreased by 80% and 5-HTExt levels by 60-80%, while evoked 5-HTExt responses are qualitatively preserved. The mice recapitulate several anomalies in putative 5-HT biomarkers reported in severe depression and exhibit depression-, anxiety-, and aggressive-like behaviors, seemingly providing a model of the behavioral alterations associated with 5-HT deficits in humans. Thus, our mutant (henceforth 5-HThypo) mice may represent a unique naturalistic model of 5-HT deficiency and, possibly, depression. Plausibly, multiple diverse insults to 5-HT homeostasis could each result in 5-HT deficiency, thus the 5-HThypo mouse likely represents a useful model of 5-HT deficiency in general as well as a model of impaired Tph2 catalytic function. The overall goal of our continued research is to use the 5-HThypo mouse to better understand how 5-HT deficiency contributes to depression etiology and affects antidepressant treatment, including the consequences for stress susceptibility and responses to current and novel therapies. For this goal, we propose four specific aims. Aim 1 will define whether 5-HT deficiency alters susceptibility to stress, as tested in the social defeat and chronic mild stress paradigms. Aim 2 will test whether antidepressant- like responses to SSRI and ketamine are affected by 5-HT deficiency. Aim 3 will pre-clinically test a novel 5- HTP-based antidepressant augmentation concept under 5-HT-deficient and normal conditions. In Aim 4 we will use conventional and state-of-the-art approaches to identify the cellular signaling pathway changes underlying the depression-like behaviors arising consequent to 5-HT deficiency. Collectively, the proposed experiments will address long outstanding questions in depression neurobiology and test a new treatment concept.